DNA-chips
In 2001, the Consortium for the Human Genome Project and the private company Celera presented the first complete example of the human genome with 30,000 genes. From this moment on, the possibility of studying the complete genome or large scale (high-throughput) studies began. So-called “DNA-chips”, also named “micro-arrays”, “DNA-arrays” or “DNA bio-chips” are apparatus that functional genomics can use for large scale studies. Functional genomics studies changes in the expression of genes due to environmental factors and to genetic characteristics of an individual. Gene sequences present small interindividual variations at one unique nucleotide called an SNP (“single nucleotide polymorphism”), which in a small percentage are involved in changes in the expression and/or function of genes that cause certain pathologies. The majority of studies which apply DNA-chips study gene expression, although chips are also used in the detection of SNPs.
The first DNA-chip was the “Southern blot” where labelled nucleic acid molecules were used to examine nucleic acid molecules attached to a solid support. The support was typically a nylon membrane.
Two breakthroughs marked the definitive beginning of DNA-chip. The use of a solid non-porous support, such as glass, enabled miniaturisation of arrays thereby allowing a large number of individual probe features to be incorporated onto the surface of the support at a density of >1,000 probes per cm2. The adaptation of semiconductor photolithographic techniques enabled the production of DNA-chips containing more than 400,000 different oligonucleotides in a region of approximately 20 μm2, so-called high density DNA-chips.
In general, a DNA-chip comprises a solid support, which contains hundreds of fragments of sequences of different genes represented in the form of DNA, cDNA or fixed oligonucleotides, attached to the solid surface in fixed positions. The supports are generally glass slides for the microscope, nylon membranes or silicon “chips”. It is important that the nucleotide sequences or probes are attached to the support in fixed positions as the robotized localisation of each probe determines the gene whose expression is being measured. DNA-chips can be classified as:                high density DNA-chips: the oligonucleotides found on the surface of the support, e.g. glass slides, have been synthesized “in situ”, by a method called photolithography.        low density DNA-chips: the oligonucleotides, cDNA or PCR amplification fragments are deposited in the form of nanodrops on the surface of the support, e.g. glass, by means of a robot that prints those DNA sequences on the support. There are very few examples of low density DNA-chips which exist: a DNA-chip to detect 5 mutations in the tyrosinase gene; a DNA-chip to detect mutations in p53 and k-ras; a DNA-chip to detect 12 mutations which cause hypertrophic cardiomypathy; a DNA-chip for genotyping of Escherichia coli strains; or DNA-chips to detect pathogens such as Cryptosporidium parvum or rotavirus.         
For genetic expression studies, probes deposited on the solid surface, e.g. glass, are hybridized to cDNAs synthesized from mRNAs extracted from a given sample. In general the cDNA has been labelled with a fluorophore. The larger the number of cDNA molecules joined to their complementary sequence in the DNA-chip, the greater the intensity of the fluorescent signal detected, typically measured with a laser. This measure is therefore a reflection of the number of mRNA molecules in the analyzed sample and consequently, a reflection of the level of expression of each gene represented in the DNA-chip.
Gene expression DNA-chips typically also contain probes for detection of expression of control genes, often referred to as “house-keeping genes”, which allow experimental results to be standardized and multiple experiments to be compared in a quantitive manner. With the DNA-chip, the levels of expression of hundreds or thousands of genes in one cell can be determined in one single experiment. cDNA of a test sample and that of a control sample can be labelled with two different fluorophores so that the same DNA-chip can be used to study differences in gene expression. DNA-chips for detection of genetic polymorphisms, changes or mutations (in general, genetic variations) in the DNA sequence, comprise a solid surface, typically glass, on which a high number of genetic sequences are deposited (the probes), complementary to the genetic variations to be studied. Using standard robotic printers to apply probes to the array a high density of individual probe features can be obtained, for example probe densities of 600 features per cm2 or more can be typically achieved. The positioning of probes on an array is precisely controlled by the printing device (robot, inkjet printer, photolithographic mask etc) and probes are aligned in a grid. The organisation of probes on the array facilitates the subsequent identification of specific probe-target interactions. Additionally it is common, but not necessary to divide the array features into smaller sectors, also grid-shaped, that are subsequently referred to as sub-arrays. Sub-arrays typically comprise 32 individual probe features although lower (e.g. 16) or higher (e.g. 64 or more) features can comprise each subarray.
One strategy used to detect genetic variations involves hybridization to sequences which specifically recognize the normal and the mutant allele in a fragment of DNA derived from a test sample. Typically, the fragment has been amplified, e.g. by using the polymerase chain reaction (PCR), and labelled e.g. with a fluorescent molecule. A laser can be used to detect bound labelled fragments on the chip and thus an individual who is homozygous for the normal allele can be specifically distinguished from heterozygous individuals (in the case of autosomal dominant conditions then these individuals are referred to as carriers) or those who are homozygous for the mutant allele.
Another strategy to detect genetic variations comprises carrying out an amplification reaction or extension reaction on the DNA-chip itself.
For differential hybridisation based methods there are a number of methods for analysing hybridization data for genotyping:                Increase in hybridization level: The hybridization level of complementary probes to the normal and mutant alleles are compared.        Decrease in hybridization level: Differences in the sequence between a control sample and a test sample can be identified by a fall in the hybridization level of the totally complementary oligonucleotides with a reference sequence. A complete loss is produced in mutant homozygous individuals while there is only 50% loss in heterozygotes. In DNA-chips for examining all the bases of a sequence of “n” nucleotides (“oligonucleotide”) of length in both strands, a minimum of “2n” oligonucleotides that overlap with the previous oligonucleotide in all the sequence except in the nucleotide are necessary. Typically the size of the oligonucleotides is about 25 nucleotides. The increased number of oligonucleotides used to reconstruct the sequence reduces errors derived from fluctuation of the hybridization level. However, the exact change in sequence cannot be identified with this method; sequencing is later necessary in order to identify the mutation.        
Where amplification or extension is carried out on the DNA-chip itself, three methods are presented by way of example:
In the Minisequencing strategy, a mutation specific primer is fixed on the slide and after an extension reaction with fluorescent dideoxynucleotides, the image of the DNA-chip is captured with a scanner.
In the Primer extension strategy, two oligonucleotides are designed for detection of the wild type and mutant sequences respectively. The extension reaction is subsequently carried out with one fluorescently labelled nucleotide and the remaining nucleotides unlabelled. In either case the starting material can be either an RNA sample or a DNA product amplified by PCR.
In the Tag arrays strategy, an extension reaction is carried out in solution with specific primers, which carry a determined 5′ sequence or “tag”. The use of DNA-chips with oligonucleotides complementary to these sequences or “tags” allows the capture of the resultant products of the extension. Examples of this include the high density DNA-chip “Flex-flex” (Affymetrix).
For genetic diagnosis, simplicity must be taken into account. The need for amplification and purification reactions presents disadvantages for the on-chip extension/amplification methods compared to the differential hybridization based methods.
Typically, DNA-chip analysis is carried out using differential hybridization techniques. However, differential hybridization does not produce as high specificity or sensitivity as methods associated with amplification on glass slides. For this reason the development of mathematical algorithms, which increase specificity and sensitivity of the hybridization methodology, are needed (Cutler D J, Zwick M E, Carrasquillo M N, Yohn C T, Tobi K P, Kashuk C, Mathews D J, Shah N, Eichler E E, Warrington J A, Chakravarti A. Geneome Research; 11:1913-1925 (2001).
The problems of existing DNA-chips in simultaneously detecting the presence or absence of a high number of genetic variations in a sensitive, specific and reproducible manner has prevented the application of DNA-chips for routine use in clinical diagnosis, of human disease. The inventors have developed a sequential method of processing and interpreting the experimental data generated by genotyping DNA-chips based on an increase in hybridization signal. The method produces high levels of specificity, sensitivity and reproducibility, which allow the DNA-chips developed on the basis of this method to be used for example, for reliable clinical genetic diagnosis.
Inflammatory Bowel Disease
Inflammatory Bowel Disease (IBD) is characterized by chronic inflammation of the intestine. This pathology presents two clinical forms, Crohns Disease (CD) and Ulcerative Colitis (UC). CD can affect any area of the intestinal tract and is associated with irregular internal injuries of the intestinal wall, while in the case of UC the inflammation is limited to the rectum and colonic mucosa and the injuries are continuous and superficial. The annual rate of UC and CD in Spain is from 4 to 5 and from 1.8 to 2.5 cases per 100,000 people, respectively. In the United States the prevalence of these diseases can reach numbers of 200 to 300 in every 100,000. The disease has a severe effect on quality of life, in particular given its chronic progress, evolution in outbreaks and frequent need for surgery. Patients of both suffer inflammation of the skin, eyes and joints.
Treatments for IBD include immunosuppressants, anti-inflammatory agents, such as antibodies targeted against tumour necrosis factor α (TNF-α) and surgery. The molecular biology of the pathogenesis of IBD is still not clear, but causative factors appear to include bacterial infection in the intestinal wall and an imbalance in the regulation of the bowel immune response.
CD and UC are classified as autoimmune diseases, both being more prevalent in individuals who have previously had another autoimmune condition. There is a predominance of CD in the female population and of UC in the male, predominantly in the older age bracket with distal proctitis or colitis.
Epidemiologic and genetic studies have provided evidence of the presence of genetic susceptibility factors for IBD, increasing expectations that the identification of genes related to IBD could bring a better understanding of the pathogenesis, diagnosis, location, and prognosis and appropriate treatment. Starting from informal studies to evaluate the risk of contracting the disease, such as segregation analysis, evidence has been provided of a genetic origin. Between 10-20% of the relatives of patients affected by CD or UC also suffered from these diseases. However, the tendency to CD and UC is complex and includes various genes as well as environmental factors. IBD is considered to be a complex genetic disease in which inheritance is not considered to be a simple Mendelian trait. Numerous studies of the association between genome and disease susceptibility have recently identified several genes in which one or more genetic variations results in a higher or lower risk of contracting the disease, a better or worse response to drugs or a better or worse prognosis.
For this reason, the clinical application of a DNA-chip to characterize the genetic variations associated with IBD will provide benefits for diagnosis and treatment. From a clinical point of view, the early diagnosis, prognosis and location of the disease would influence therapeutic decisions as to treatment of IBD. At least two different groups would benefit from this development:                relatives of IBD patients who are interested in knowing their likelihood of developing the disease; and        patients who have IBD, in order to be able to choose a personalised therapy, depending on the risk of inflammation or fistulae. The higher the risk of contracting a severe form of IBD, the greater the need for more aggressive therapy.        
Apart from the contribution to diagnosis and treatment of IBD and the development of new therapeutic strategies, progress in the physiopathology of the inflammatory reaction in IBD will also be of interest in the study of a wide range of autoimmune diseases including several neurodegenerative diseases, rheumatoid arthritis and dermatological conditions such as psoriasis.
A DNA-chip, which allows the simultaneous, sensitive, specific and reproducible detection of genetic variations associated with IBD, could be used clinically in diagnosing IBD.
Erythrocyte Antigens
The blood of each person is so characteristic that it can serve as a means of identification that is nearly as precise as fingerprints; only identical twins have exactly the same blood characteristics. Blood group determination is particularly useful in medical fields such as blood transfusions, haemolytic diseases in fetuses and the new born, medical-legal applications and organ transplantation.
The majority of transfusions can be considered safe. However, sometimes they produce slight reactions or possibly a serious and even fatal reaction. Temperature and allergic (hypersensitivity) reactions, occur in 1-2% of transfusions, but more serious incompatibilities do exist which cause the destruction of red cells, (a haemolytic intravascular reaction).
Foetal and new born haemolytic disease (HDNF) is a well known immunological condition, in which the potential for survival of the fetus or new born is compromised due to the action of maternal antibodies that pass through the placenta and specifically target antigens of paternal origin present in the red cells of the fetus or new born. It has been determined that EHPN is not only due to antibodies against the D antigen, but that antigens of the RH system, the ABO system and others are also involved.
Correct genotyping of blood groups therefore has importance in transfusions (including the detection of rare or infrequent alleles).
Blood groups are composed of alloantigens present on the surface of the erythrocyte membrane and red cells, which are transmitted from parents to children according to the laws of Mendelian genetics.
The International Society of Blood Transfusions has classified more than 26 different human blood groups. The majority have been defined at a genetic level and include polymorphisms at one unique nucleotide (SNPs), genetic deletions, conversions and other events, which result in genetic variation. The blood group antigens can be classified in two large groups:                A. Antigens determined by carbohydrates.        B. Antigens determined by proteins.A. Antigens Determined by CarbohydratesGroup ABO        
This blood group is of clinical importance because it causes the majority of incompatibility reactions in transfusions and organ transplants. The biochemical basis of group ABO depends on the activity of an N-acetylgalactosamine transferase in individuals of blood group A and a galactosyl transferase in blood group B; whilst individuals belonging to group O lack an active transferase enzyme. The genetic basis of the ABO phenotypes is the substitution of amino acids in the ABO gene of glycosyltransferase. This gene is 19,514 bases in size and encodes a membrane bound enzyme that uses GalNAc or UDP-Gal as a substrate. Four amino acid changes in exons 6 and 7 of the ABO gene are responsible for substrate specificity of the transferases A and B respectively, within them the changes Gly235Ser and Leu266Met are vital. The majority of individuals of group O present deletion of one single nucleotide (A261G) which gives rise to a change in the reading frame and results in the production of an inactive transferase protein. Nonetheless, a growing number of O alleles (about 20) exist that result in nonexpression of the transferases A or B. Rare alleles of the subgroup ABO, like A3, Ax, Ael, B3Bx and Bel have been described. These alleles have arisen from genetic recombinations from different alleles of the ABO group.
B. Antigens Determined by Proteins.
B.1. Antigens Dependent on Expression of Erythrocyte Transferase Molecules.
Rh (RH)
Incompatibility of RH occurs in a large portion of transfusion reactions and is the main cause of hemolytic disease in newborn and fetuses (HDNF). The RH antigens come from two proteins (RH CcEe and RH D) encoded by the RH locus (1p34-36.2) that contains the genes RHD and RHCE (70 Kb). Possibly the positive D haplotypes present ay configuration of the genes RHD-RHCE of the same orientation, while the negative D haplotypes present a reverse orientation. The negative D phenotype, common in old European populations, is caused by a deletion of the gene RHD. This seems to have been generated by an unequal crossing over between the genes RHCE and RHD. In the African population a pseudogene of RHD is the predominant D negative allele but its frequency diminishes amongst Afro-Americans and Afro-Caribbeans. Recombinations between the genes RHCE and RHD cause rare hybrids that lead to a partial expression of the D antigen. These uncommon antigens on some occasions have been identified as clinically significant.
The proteins RH CcEe and RH D co-express themselves with an equivalent glycoprotein (36% identity), the associated glycoprotein RH (RHAG). This erythrocyte specific complex is possibly a hetero tetramer implicated in bidirectional ammonia transport. The mutations in RHAG are the causes of RH null syndrome, associated with defects in transport across the erythrocyte membrane, deficiencies in CD47 and a total absence of ICAM-4. Furthermore, genes related to RHAG, RHBG and RHCG have been found in the regions 1q21.3 and 15q25 respectively. These genes are expressed in different forms in different human tissue.
Kidd (JK)
The Kidd (JK) antigens occur in the urea transporter hUT-B1 of red cells. The significance of the Kidd antigen has been known for two decades when it was discovered that JK (a−b−) red cells were resistant to lysis in 2M urea. The molecular basis of the expression of the Kidd antigen is a SNP in nucleotide 838 (G-A) causing a change Asp280Asn (JK*A-JK*B). The Kidd null phenotype, JK (a−b−) is due to mutations causing fame-shift mutations, premature termination of translation, inappropriate gene splicing and partial deletions in the gene SLC14A1.
Diego (DI)
The antigens of the blood group Diego (DI) are the most abundant proteins on the surface of red cells (1.1 million copies per cell), and are crucial for carrying CO2 and acid-base homeostasis. It is thought that Di antigens vary due to multiple SNPs present in the gene SLC4A1.
Colton (CO)
The CO antigens (COa, COb and CO3) are expressed by the carrier molecule AQP-1. The (COa-COb) antigens are produced by a SNP in AQP-1 that produces a change in codon 45 from alanine to valine.
B.2 Antigens Determined by Expression of Red Cell Membrane Enzymes.
Kell (KEL)
The antigens of the KEL system are very important in transfusions; the k antigen is the second main cause of haemolytic disease in the new born. The glycoprotein KEL is a type II membrane protein. The C-terminal catalytic regions process large endothelins that are potent vasocontrictors. Cysteine 72 of the glycoprotein KEL forms a disulphide bridge with the protein Kx, which might explain why erythrocytes null for KEL (Ko) show activation of levels of the Kx antigen. The antigen of this system with most clinical importance, K (KEL1), is associated with a change Met193Thr that allows Asn-X.ThrN-glycosylation to occur.
Dombrock (DO)
The variants DOa/DOb are due to an SNP in the gene DOK1, which encodes an enzyme ADP ribosyltransferase, that affects codon 265 (Asn-Asp). The ADP ribosyltransferase of red cells could help eliminate the NAD+ of serum, but it has been noted that it also takes part in the post-transcriptional modification of other proteins. The RGD motif and DOb take part in cellular adhesion. Oddly the allelic variant DO*B is more common in African and Asian populations and could be an evolutionary advantage against the invasion of Plasmodium falciparum which expresses RGD proteins during its infection process.
B.3. Antigens Determined by Expression of Membrane Receptors of Red Cells.
Duffy (FY)
The function of the glycoprotein FY as a cytokine receptor of red cells is to accelerate proinflammatory cytokine signalling. The FY glycoprotein is the erythrocyte receptor for the malarial parasite Plasmodium vivax and as a consequence FY negative individuals (FY a-b-) are very common in populations where this parasite is found (Western Africa). Three main alleles of FY exist: FY*A, FY*B and FY*A and B which differ due to an SNP which alters codon 42, while phenotype FY (a−b−) in Africans is caused by a SNP (C-T) in the FY gene promoter that results in an absence of FY glycoprotein in the erythrocytes.
MNSs (MNS)
The MNS antigens are generated against glycoporin A, while the Ss antigens are against glycoporin B. The genes GYPA and GYPB line up in tandem in the locus 4q28-31 but there is no relationship between glycoporins C and D. Two amino acid changes in the N-terminal region of GPA are responsible for the blood group M-N and a change in amino acid in GPB determines the blood group S-s. A large number of MNS alleles exist due to genetic recombinations, genetic conversions or SNPs.
Human blood groups have been defined at a genetic level for the majority of antigens with clinical significance. Nevertheless, genotyping of red cells is still only performed rarely, mainly in prenatal determination of blood groups in cases of haemolytic diseases in newborns and fetuses.
The compatibility of blood transfusions between donors and recipients is generally evaluated by serological techniques (antibody-antigen reactions). The use of these techniques can give incorrect results, which could lead to a potential adverse immune reaction in the recipient (patient). No serological tests exist for a high number of the so-called ‘weak’ genes and on various occasions the antibodies used have not been sufficiently specific. The only process capable of preventing problems of this type is that based on complete molecular genotyping of both the donor and the recipient.
SNP genotyping will allow both these determinations to be carried out on a large scale and also the genotyping of rare alleles in blood groups that with existing techniques cannot be determined.
The appearance of new alleles in certain blood groups (e.g. RH) will continue and will therefore require technology capable of progressing and being constantly monitored. The Human Genome project has identified new SNPs in many proteins in the blood groups concerned, although it still needs to be serologically determined if these SNPs are in antigens related to blood groups.
Nowadays genetic molecular analysis is common in transfusions. For example, detection of viral contamination, such as the hepatitis C virus (HCV), the human immunodeficiency virus (HIV) or the hepatitis B virus (HBV), by PCR methodology from small volumes of plasma has been common practice in the European Union (EU) since 1999. Diagnosis based on PCR has practically taken the place of serology in the determination of HLA (human leukocyte antigen); and is routinely used in transfusion centres involved in bone marrow transplants.
One of the discoveries of the Human Genome project was the high frequency of polymorphisms in a single nucleotide (SNPs) found in human DNA. Approximately one SNP was found for every kilobase. This discovery has pushed forward the technical development of rapid diagnosis of SNP genotyping, for example by using DNA-chips. This new technology can be applied to developing a rapid method of genotyping of blood groups.
Diverse methods of diagnosis for different blood groups have been described. As an illustrative example, U.S. Pat. No. 5,80,4379 relates to a molecular method of diagnosis and a kit to determine the genotypes of the blood group KEL. U.S. Pat. No. 5,723,293 relates to a method and kit to determine the genotypes of the blood group RH. Furthermore a serological diagnostic test to classify blood groups from blood or serum has been described. Likewise new genetic variations of the blood group Duffy have been described as a method of genotyping this blood group.
However, no method has been described based on DNA-chip technology capable of being an open platform for genotyping of all the allelic variants of the blood groups with major clinical relevance (including rare variants) that can be used as a method of diagnosis on a huge scale in the population.
A DNA-chip which allows the simultaneous, sensitive, specific and reproducible detection of genetic variations associated with determined erythrocyte antigens could be used clinically for genotyping antigens of blood erythrocytes on a large scale in the population and therefore for determining blood groups in humans.
Adverse Reactions to Medicine
Any medicine is developed with the intention of curing, relieving, preventing or diagnosing an illness or disease but unfortunately these can also produce adverse effects with a risk, which, depending on the specific case, could range from minimal to severe. Although difficult to calculate, the risk of the treatment should not be ignored and the order of magnitude should be known by the doctor and also the patient and accepted, with the understanding that the potential benefit of the medicine compensates any of these risks.
An adverse reaction is any harmful or unwanted effect that happens after the administration of the dose usually prescribed to a human being for the prophylaxis, diagnosis or treatment of a disease. Present consensus allows this definition, which was created by the World Health Association in 1972, to be understood in the following manner: “It is any unwanted effect that appears on administering a medicine of adequate dose, for the prophylaxis, diagnosis or treatment of a disease or for the modification of a physiological function.”
Developed countries count on systems of drug vigilance to centralize the supervision of security and efficiency of drugs used, which are responsible for collecting and analyzing details of adverse reactions suspected of being produced by the drug used on the market.
In Spain the first steps in creating a system of pharmacovigilance were started in the 70s and in 1983, Spain incorporated the International Programme of Pharmacovigilance of Health. In 1992 a computerized database called FEDRA (Spanish Pharmacovigilance of Data of Adverse Reactions) was created. The pharmaceutical industry actively collaborates with this system, and moreover as established by The 1986 General Health Act, and also The 1990 Medicine Act, all public health personnel, including doctors, pharmacists, vets and nurses, are obliged to notify health authorities of any suspicion of adverse reactions to drugs known to them and to collaborate with the Spanish system of pharmacovigilance. Spain also collaborates with the European Medical Evaluation Agency which came into operation in 1995. From the information collected by FEDRA it appears that Spain is within the group of countries with the highest rate of notification, with an average similar to Germany and France although lower than countries such as the USA, Ireland, Norway, New Zealand, The UK or Sweden.
Nowadays, in countries like Spain, where the older population is growing and more medicine is being administered, particularly to this age group and also with increasing self-medication, it is only to be expected that the problem of adverse reactions may be important. The Centre for Drug Evaluation and Research of the FDA (U.S. Food and Drug Administration), confirms that more than two million adverse reactions occur annually in the USA, which cause about 100,000 deaths a year, being the fourth cause of death ahead of lung disease, diabetes, AIDS, pneumonia and traffic accidents. The number of patients that die in England and Wales due to errors
in prescription of medicines or adverse reactions is growing and the difficulty is that the extent of the problem is not known. In Spain, five out of every hundred casualty cases in public hospitals are due to adverse reactions to drugs and between 10-20% of those hospitalized suffered this medical mishap on receiving medication. Of those affected, 1% die as a consequence.
Until May 2000 about 80,000 notifications of adverse reactions to registered drugs had been recorded in the database at the Centre for Pharmaceutical Vigilance in Catalunya. Of these, two thirds were spontaneous and came from primary care. Of those reactions notified most were minor or moderate, whilst 12% were serious and 1% fatal. 50% of reactions were skin, digestive or neurological. The majority of decisions to withdraw drugs are related to hepatic/liver and haematological reactions. What causes concern is that these types of reactions, which represent a small percentage of the total, are those where the majority of drugs are withdrawn. Antibiotics are the main cause of adverse effects, followed by anti-rheumatic drugs and painkillers and drugs to prevent cardiovascular disease. The detection of adverse effects can provoke not only the withdrawal but also the decision to change the use of the drug, or the reformulation or introduction of new directions for specific patients.
A DNA-chip, which allows the simultaneous, sensitive, specific and reproducible detection of genetic variations associated with adverse reactions to medicine, could be clinically useful to prevent or reduce the aforementioned reactions in patients receiving medical treatment.